Cell collection apparatus, cell collecting system, and cell collecting method

ABSTRACT

In order to pick up a sufficient amount of cells for a genetic test from a section of a biological tissue with a simple structure and a simple operation, provided is a cell collection apparatus comprising: a substrate which is provided to be dividable into a plurality of small pieces along a predetermined dividing line, and which has a flat surface to which a section of a biological tissue can be pasted; a sheet-shaped expandable member to which the substrate can be adhered in a detachable manner, and which is expandable in a direction along the surface; a expansion unit for expanding the expandable member in at least a region adhered with the substrate, in a direction along the surface; and a pickup unit for detaching and picking up the divided small pieces from the expandable member.

TECHNICAL FIELD

The present invention relates to a cell collection apparatus, a cellcollecting system, and a cell collecting method.

BACKGROUND ART

Heretofore, the Laser Microdissection (LMD) method has been known as atechnique to pick up a micro-region of about several tens microns bycutting it out from a thin tissue section for use in pathologicaldiagnosis and the like. In the LMD method, the micro-region of thetissue section to be picked up is irradiated with UV laser, by which themicro-region is cut out from the section.

CITATION LIST Non Patent Literature {NPL 1}

-   Leica MICROSYSTEMS, “Leica LMD 7000”, [online] [searched on Apr. 23,    2010], internet    <URL:http://www.leica-microsystems.co.jp/Website/Products.nsf/(ALLIDs)/9482D2B75AA53E5F49257555001FB123>

SUMMARY OF INVENTION Technical Problem

However, in the case where a genetic test is desired to be conductedwith use of a gene extracted from cells contained in a minute fragmentthat has been prepared by cutting out a region where specific cells suchas cancer cells exist, from a section; it is not possible to extract asufficient amount of the gene necessary for the test, because the numberof cells contained in the fragment having been picked up by the LMDmethod is too small. Accordingly, a large number of sections have to besubject to microdissection to collect a sufficient number of cells.Therefore, large amounts of labor and time are required for conductingone genetic test.

On the other hand, it can be considered to increase the area of thefragment to be cut out, or to increase the thickness of the section tobe cut out from the biological tissue. However, if the area of thefragment cut out by the LMD method is increased, it is necessary toincrease the size of the laser-scannable region, or to increase thelaser output so that the fragment having been cut out by laser scanningcan be blown off by the pressure of laser. Therefore, the scale of thedevice structure has to be much enlarged and also the price of thedevice has to be raised. In addition, if the thickness of the section isincreased, the disadvantage is that it is necessary to increase thelaser output after all so as to cut out the section by laser.

The present invention addresses the above-mentioned situation. It is anobject of the present invention to provide a cell collection apparatus,a cell collecting system, and a cell collecting method, capable ofpicking up a sufficient amount of cells for a genetic test from asection of a biological tissue with a simple structure and a simpleoperation.

Solution to Problem

A first aspect of the present invention is a cell collection apparatuscomprising: a substrate which is provided to be dividable into aplurality of small pieces along a predetermined dividing line, and whichhas a flat surface to which a section of a biological tissue can bepasted; a sheet-shaped expandable member to which the substrate can beadhered in a detachable manner, and which is expandable in a directionalong the surface; a expansion unit for expanding the expandable memberin at least a region adhered with the substrate, in a direction alongthe surface; and a pickup unit for detaching and picking up the dividedsmall pieces from the expandable member.

In the first aspect, the expandable member may have stickiness on itssurface.

Moreover, in the first aspect, the substrate may comprise a plurality ofseparated small pieces adhered to the expandable member in mutuallyadjacent and aligned state.

Moreover, in the first aspect, the dividing line may be composed of agroove formed in the surface of the substrate.

Moreover, in the first aspect, the expandable member may comprise anoptically transparent or semi-transparent material.

Moreover, in the first aspect, the small piece may be in a cuboid-shapehaving a thickness of 0.05 to 0.5 mm and side lengths of 0.05 to 5.0 mm.

Moreover, in the first aspect, the expansion unit may comprise: a fixingmember for fixing a periphery of the region of the expandable memberbeing adhered with the substrate, in a predetermined shape; and apressing member for pressing the region of the expandable member beingfixed by the fixing member, from a side of the surface opposite side tothe surface adhered with the substrate.

Moreover, in the first aspect, the pickup unit may also comprise: aneedle member for pushing a position of the expandable member beingadhered with the small pieces, from the surface on the opposite side tothe surface adhered with the small pieces.

Moreover, in the first aspect, the structure may also comprise: aholding member for holding the region of the expandable member in aexpanded state.

Further, in the above-mentioned structure, the holding member may becapable of holding the surface of the expandable member on the sideadhered with the substrate to be faced downward.

Moreover, the structure comprising the holding member may also comprise:a suction member which is provided to the side of the surface of theexpandable member held by the holding member where the substrate isadhered, and the interior of which can be sucked to a negative pressure.

Moreover, in the first aspect, the small piece may be a magneticparticle having a diameter of 0.001 to 0.5 mm.

Moreover, in the above-mentioned structure, it is preferable that thepickup unit has a magnet which generates a magnetic force to attract themagnetic particle, in a space of an approximately same size as that ofthe dimension of the small piece.

Moreover, a second aspect of the present invention is a cell collectingsystem comprising: a cell collection apparatus according to any one ofthe above-mentioned structure; and an observation device for observingthe section on the substrate being adhered to the expandable member.

Further, a third aspect of the present invention is a cell collectingmethod comprising: a pasting step for pasting a section of a biologicaltissue on a surface of a substrate that can be divided into a pluralityof small pieces along a predetermined dividing line, while having thesection bridged over the dividing line; a dividing step for dividing thesubstrate and the section of the biological tissue along the dividingline, by expanding the substrate pasted with the section in a directionalong the surface; and a pickup step for picking up the small pieces ofthe section that has been divided in the dividing step.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the overall structure of the cell collecting systemaccording to one embodiment of the present invention.

FIG. 2A is a side view of the glass substrate adhered to the stickysheet, showing a case where the grooves are faced to the sticky sheetside.

FIG. 2B is a side view of the glass substrate adhered to the stickysheet, showing a case where the grooves are faced to the opposite sideagainst the sticky sheet.

FIG. 3A shows the glass substrate adhered to the sticky sheet.

FIG. 3B shows the glass substrate divided by expanding the sticky sheet.

FIG. 4 shows the structure of the expanding stage and the jig.

FIG. 5A is a diagram explaining a method how to use the expanding stageand the jig of FIG. 4, and showing a state where the sticky sheet hasnot been expanded yet.

FIG. 5B is a diagram explaining the method how to use the expandingstage and the jig of FIG. 4, and showing a state where the sticky sheethas been expanded.

FIG. 5C is a diagram explaining the method how to use the expandingstage and the jig of FIG. 4, and showing a state where the sticky sheetis held by the grip rings.

FIG. 6 is a flowchart explaining the procedure to pick up cells from thesection with use of the cell collection apparatus and the cellcollecting system of FIG. 1.

FIG. 7 shows a modified example of the substrate.

FIG. 8 shows another modified example of the substrate.

FIG. 9 shows a modified example of the cell collecting system of FIG. 1.

FIG. 10 shows another modified example of the cell collecting system ofFIG. 1.

FIG. 11 is a flowchart showing a modified example the procedure to pickup cells with use of the cell collecting system of FIG. 1.

FIG. 12 is a diagram showing a modified example of the method to expandthe sticky sheet with use of the outer cylinder and the inner cylinder,and showing a state where the sticky sheet is set on the outer cylinder.

FIG. 13 shows a state where the sticky sheet is being expanded by usingthe outer cylinder and the inner cylinder of FIG. 12.

FIG. 14 shows a state where the sticky sheet is being expanded byanother method using the outer cylinder and the inner cylinder of FIG.12.

FIG. 15A shows another modified example of the method to expand thesticky sheet before expanding the sticky sheet.

FIG. 15B shows the another modified example of the method to expand thesticky sheet after expanding the sticky sheet.

FIG. 16 shows a modified example of the grip rings.

FIG. 17 is a photograph of the section made by the Example of thepresent invention, showing a state before the section has been divided.

FIG. 18 is a photograph showing a state after the section of FIG. 17 hasbeen divided.

FIG. 19 shows an example of an embodiment for detaching and picking upsmall pieces from the expanded sticky sheet.

FIG. 20A shows an example of an embodiment for detaching and picking upsmall pieces from the expanded sticky sheet by using a suction member.

FIG. 20B is an enlarged diagram showing the example of the embodimentfor detaching and picking up a small piece from the expanded stickysheet by using the suction member.

FIG. 21A shows an embodiment for picking up small pieces by a needlewhile tilting the expanded sticky sheet.

FIG. 21B shows an embodiment for picking up small pieces by a suctionmember while tilting the expanded sticky sheet.

DESCRIPTION OF EMBODIMENTS

Hereunder is a description of a cell collection apparatus 1 and a cellcollecting system 100 according to one embodiment of the presentinvention with reference to the drawings.

As shown in FIG. 1, the cell collecting system 100 of this embodimentcomprises an optical microscope (observation device) 2 and the cellcollection apparatus 1 of this embodiment.

The optical microscope 2 is of an erecting type. The optical microscope2 is configured such that an imaging device 3 like a CCD camera fortaking an image of the field of view is connected to a camera port 2 aso that the image taken by the imaging device 3 can be displayed on amonitor (not shown). The structure may also be such that the image takenby the imaging device 3 is subject to image processing with an imageprocessing device (not shown) like an image processor and thereafterdisplayed on the monitor.

The cell collection apparatus 1 comprises: a glass substrate (substrate)4 to which a section of a biological tissue can be pasted; a stickysheet (expandable member) 5 to which the glass substrate 4 can beadhered, and which is expandable in directions along the surface; aexpanding stage (expansion unit, pressing member) 6 for expanding thesticky sheet 5; a jig 7 for holding the sticky sheet 5 when the sheet 5is being expanded by the expanding stage 6; and a pickup unit 8 forpicking up small pieces 4 b of the glass substrate 4 from the stickysheet 5. By so doing, it is possible to divide the section pasted on thesubstrate together with the substrate along the predetermined dividingline, by expanding the expandable member with the expansion unit in astate where the substrate pasted with the section of the biologicaltissue is being adhered onto the expandable member. Accordingly, it ispossible, by picking up small pieces adhered with section fragmentscontaining the cells of interest, among the plurality of divided smallpieces, by using the pickup unit, to pick up these cells from thesection, with a simple structure and a simple operation only.

In this case, a sufficient amount of cells for a genetic test can beeasily picked up only by picking up one or a several number of cells, byincreasing the thickness of the section or enlarging the area of thesmall pieces in the planar direction so as to increase the amount ofcells adhered to each small piece.

The glass substrate 4 is shaped like a plate having a flat surface. Asshown in FIG. 2A and FIG. 3A, the surface of the glass substrate 4 isformed with grid-like grooves (dividing lines) 4 a with predeterminedspacing. The grooves 4 a can be formed by, for example, laserprocessing, chemical etching, dicing, or the like. It is also possibleto form them by hand work of an operator using a glass cutter or thelike.

By so doing, the substrate can be easily divided along the position ofthe groove when the expandable member is expanded.

Preferably, the glass substrate 4 has a thickness which enables tostably support a section of a biological tissue, for example, from 0.05to 0.5 mm. The spacing between the grooves 4 a can be modifiedappropriately according to the size of fragments to be picked up fromthe section, although it is preferably from 0.05 to 5.0 mm so that asufficient amount of cells can be contained in the picked up fragmentswhile the fragments of a desired region can be picked up from thesection with adequately precise positional accuracy. An opticallytransparent substrate is used for the glass substrate 4, so that thetransmitted beam image of the section pasted thereon can be observedwith the optical microscope 2.

By so doing, the positions of the small pieces on the expandable membercan be checked at the time when observing the transmitted beam image ofthe expandable member with the optical microscope.

Moreover, it is also possible to apply the glass substrate 4 withsilanization or such a chemical treatment on the surface so as toimprove the adhesiveness between the surface and the section. Also, itis possible to apply, for example, a coating treatment for preventingadsorption of nucleic acids, proteins, and the like, according to thepurpose of the test to be conducted after picking up the small pieces 4b.

One surface of the sticky sheet 5 is coated with an adhesive agent tohave a stickiness which enables the glass substrate 4 to be sufficientlyand firmly adhered thereto, as well as enabling it to be detachedtherefrom by a pickup unit 8 that will be described later. By so doing,the substrate can be easily adhered onto the sticky sheet. In addition,the sticky sheet 5 is expandable in directions along the surface. Whenthe sticky sheet 5 is expanded in a direction along the surface in astate where the glass substrate 4 is being adhered onto the sticky sheet5, the glass substrate 4 is also extended in the direction along thesurface together with the expanding of the sticky sheet 5, by which asshown in FIG. 3B the glass substrate 4 is cut along the positions of thegrooves 4 a and divided into a plurality of small pieces 4 b. Moreover,at this time, the section A having being pasted on the surface of theglass substrate 4 is also extended in the direction along the surface,and thus cut along the grooves 4 b.

The sticky sheet 5 is optically permeable or semi-permeable, by which anobject arranged on the opposite side to the object lens of the opticalmicroscope 2 can be observed across the sticky sheet 5, at the time whenobserving the sticky sheet 5 with the optical microscope 2. As for thesticky sheet 5, it is possible to suitably use a dicing tape for use intemporal fixation of a wafer during a dicing step in a semiconductorproduction process, for example.

Note that FIG. 2A shows an example in which the glass substrate 4 isadhered to the sticky sheet 5 while the grooves 4 a are facing to thesticky sheet 5 side. However, the structure may also be such that asshown in FIG. 2B the glass substrate 4 is adhered to the sticky sheet 5while the grooves 4 a are facing to the opposite side against the stickysheet 5.

As shown in FIG. 4, the expanding stage 6 is formed to have anapproximately columnar shape with approximately parallel and flatopposite ends. In the expanding stage 6, a step is formed on the lateralsurface so that the upper end portion (hereunder, referred to as thesmall diameter portion) 6 a has a diameter smaller than that of theother portion (hereunder, referred to as the large diameter portion) 6b.

The jig 7 has a bottom frame (fixing member) 9 a to which the stickysheet 5 can be pasted in an extended state, a top frame 9 b for coveringthe bottom frame 9 a from the top, and grip rings 10 a and 10 b (holdingmember) for holding the sticky sheet 5 in a expanded state. By so doing,the expandable member can be expanded to be held in a state where thespacing between the respective small pieces is kept open. This makes itmuch easier to pick up the small pieces with the pickup unit. The griprings 10 a and 10 b consist of an outer ring 10 a and an inner ring 10b. The inner diameter of the outer ring 10 a and the outer diameter ofthe inner ring 10 b are formed to be approximately the same. Inapproximately the center of the bottom frame 9 a and the top frame 9 b,windows 9 c and 9 d passing through in the plate thickness direction arerespectively formed. The outer ring 10 a is fit inside the window 9 d ofthe top frame 9 b, along the inner circumferential surface thereof. Thewindow 9 c of the bottom frame 9 a has the same or larger diameter thanthe outer diameter of the small diameter portion 6 a.

The jig 7 is used in the following manner. Firstly, the inner ring 10 bis fit along the outer circumference of the small diameter portion 6 a.Then, the sticky sheet 5 adhered with the glass substrate 4 is adheredto the bottom surface of the bottom frame 9 a in a state where the glasssubstrate 4 is arranged in a position of the window 9 c and the surfaceon the side adhered with the glass substrate 4 is facing upward. By sodoing, as shown in FIG. 5A, the periphery around the region adhered withthe glass substrate 4 of the sticky sheet 5 is fixed in a predeterminedshape. Next, the bottom frame 9 a is pressed to move downward in a statewhere a portion of the sticky sheet 5 corresponding to the window 9 c isbeing pressed against the top surface of the expanding stage 6. By sodoing, as shown in FIG. 5B, the portion of the sticky sheet 5corresponding to the window 9 c is expanded by being pressed from theexpanding stage 6, and the glass substrate 4 is divided into a pluralityof small pieces 4 b together with the expanding of the sticky sheet 5.

Next, as shown in FIG. 5C, the outer ring 10 a and the inner ring 10 bare fit to each other by covering the top frame 9 b over the bottomframe 9 a in a state where the sticky sheet 5 is being expanded. By sodoing, the portion of the sticky sheet 5 corresponding to the window 9 cis held in a expanded state by the grip rings 10 a and 10 b.

It is also possible to put a mark, for example, a scale mark, on thesticky sheet 5 for the purpose of understanding the degree of how muchthe sticky sheet 5 is expanded when the sticky sheet 5 is being expandedin this manner.

The pickup unit 8 comprises a pickup needle 11 and a manipulator 12 foroperating the pickup needle 11. The tip of the pickup needle 11 has adiameter approximately the same as or smaller than the size of eachsmall piece 4 b. The manipulator 12 holds the pickup needle 11 at thedistal end of the arm 12 a. The manipulator 12 is designed to move thearm 12 a in three dimensional directions by the operation of theoperator.

By so doing, only specific small pieces can be easily detached andpicked up, among the plurality of small pieces, from the expandablemember.

Hereunder is a description of the method to pick up fragments containingcells of interest from a section of a biological tissue by using thethus configured cell collection apparatus 1 and cell collecting system100, with reference to FIG. 6.

In order to pick up the target cells from a section of a biologicaltissue by using the cell collection apparatus 1 and the cell collectingsystem 100 according to this embodiment: firstly, the section forpick-up purpose (section) is cut out from the biological tissue (StepS1); and the thus cut out section for pick-up purpose is pasted on theglass substrate 4 (Step S2, Pasting Step). Then, the analyte sectionimage is acquired by observing the section for pick-up purpose with theoptical microscope 2 (Step S3).

The section for pick-up purpose is cut out by, for example, freezesectioning or paraffin sectioning. The thickness of the section forpick-up purpose may be thicker than the thickness of a usual section foruse as a pathological sample or the like (about several to tenmicrometers), although it depends on the purpose of the application ofthe picked up cells. For example, the section for pick-up purpose can becut out to have a thickness of about 50 μm. If the size of the thus cutout section for pick-up purpose is smaller than the spacing between thegrooves 4 a of the glass substrate 4, the section for pick-up purpose ispasted on the glass substrate 4 so that the section for pick-up purposecan be bridged over the groove 4 a.

Moreover, differently from the section for pick-up purpose, a sectionfor staining purpose is cut to have a thickness of about several to tenmicrometers from the place adjacent to the place where the section forpick-up purpose of the biological tissue has been cut out. The thus cutout section for staining purpose is pasted on a slide glass and thenstained with, for example, a dye for pathological diagnosis (Step S4).Then, the stained-section image is acquired by observing the stainedsection for staining purpose with the optical microscope 2 (Step S5). Inthe acquired stained-section image, the region to be picked up where thecells of interest exist, for example, a region infiltrated by cancercells, is determined (Step S6).

Next, the acquired analyte section image and the stained-section imageare superposed and displayed so that the sections in the respectiveimages can exactly overlap each other (Step S7), and the positions ofthe small pieces 4 b corresponding to the determined region to be pickedup in the section for staining purpose, for example, the column numbersand the row numbers of the small pieces 4 b, are recorded (Step S8).

Next, the glass substrate 4 is adhered to the sticky sheet 5. It is alsopossible to paste the cut out section for pick-up purpose, onto theglass substrate 4 that has been previously adhered to the sticky sheet5. Next, the sticky sheet 5 is expanded by using the expanding stage 6and the jig 7, to thereby divide the glass substrate 4 (Step S9,Dividing Step). At this time, the section for pick-up purpose pasted onthe glass substrate 4 is also extended together with the glass substrate4 in the planar direction, by which the section is divided intofragments along the positions of the grooves 4 a.

Next, the sticky sheet 5 is held in a expanded state by the grip rings10 a and 10 b, and the sticky sheet 5 is set on the specimen stage 2 bso that the surface on the side adhered with the glass substrate 4 isfaced downward. At this time, it is either possible to set the stickysheet 5 on the specimen stage 2 b in a state detached from the bottomframe 9 a, or to set the sticky sheet 5 on the specimen stage 2 btogether with the bottom frame 9 a.

Next, while observing the small pieces 4 b in the recorded positionswith the optical microscope 2, the manipulator 12 is operated within thefield of view thereof to arrange the pickup needle 11 on the back of asmall piece 4 b to be picked up. The back face of the small piece 4 b tobe picked up is pushed with the pickup needle 11 to thereby detach anddrop the small piece 4 b from the sticky sheet 5 (Step S10, PickupStep). At this time, it is also possible to arrange a tube rack 13 underthe specimen stage 2 b so that collection tubes 13 a can be previouslyarranged in the positions where the small pieces 4 b would drop.

By so doing, it is possible to detach the small pieces in a state wherethe sticky sheet is held in a state where the substrate is facingdownward, by using the grip rings. This makes it possible to easilycollect the small pieces by letting them drop by their own weights.

Throughout the above-mentioned procedure, fragments of the region wherethe cells of interest exist can be picked up from the section forpick-up purpose, together with the small pieces 4 b. The picked up cellsare used in, for example, a genetic test or the like.

In other words, the selection of small pieces to be picked up and thepickup of the selected small pieces can be more easily and accuratelyconducted, through observation of the section pasted on the substrate,the small pieces formed after dividing the substrate, and the like, withthe optical microscope.

In this way, according to this embodiment, the advantage is that theregion where the cells of interest exist can be selectively picked upfrom the section with a simple structure and a simple operation only. Inaddition, since there is no need of an expensive structure such as a UVlaser light source, another advantage is that the production cost can bekept low. Moreover, unlike the LMD, the section is mechanically cut.Thus, a section having a relatively thick thickness can be easily cut,and also the sizes of the small pieces 4 b can be easily enlarged onlyby adjusting the spacing between the grooves 4 a. Accordingly, theoperation that has so far required a large amount of labor becausefragments have to be picked up from a plurality of sections so as tocollect a sufficient number of cells, can be sufficed only by picking upone or a small number of small pieces 4 b. Thus, the advantage is thatthe labor and the time required for the operation can be remarkablyreduced.

In the above-mentioned embodiment, the glass substrate 4 formed with thegrid-like grooves 4 a is used as the substrate for pasting the section.However, instead of this, it is also possible to use a glass substrate 4comprising glass-made small pieces 4 b aligned in the planar direction.

In this case, in order to keep the shape of the glass substrate 4, theglass-made small pieces 4 b are previously adhered onto the sticky sheet5 in an aligned state as shown in FIG. 7. By so doing, the glasssubstrate 4 can be more easily and reliably divided along the border(dividing lines) between the small pieces 4 b when the sticky sheet 5 isexpanded.

The following method can be adopted as an example to produce the alignedstate of the previously divided small pieces on the sticky sheet 5.

The glass substrate 4 is adhered on the sticky sheet 5, and the glasssubstrate 4 is scratched by a glass cutter. Then, the glass substrate 4is cracked along the position of the scratch by hand. By so doing, thealigned state of the small pieces 4 b on the sticky sheet 5 as shown inFIG. 7 can be easily produced. At this time, it is preferable to carryout this procedure while protecting the surface of the glass substrate 4with a film or the like so as to avoid contamination of the glasssubstrate 4.

In addition, the structure of the substrate employed in theabove-mentioned embodiment is only an example, and the present inventionis not to be limited to this structure.

For example, the material of the substrate may be not only a glass butalso a resin or the like. Regarding the shape of the small piece 4 b,any shape may be adopted as long as the small pieces 4 b can be denselyaligned on the surface of the sticky sheet 5. For example, a regularhexagonal shape or a triangular shape may be adopted. Moreover, as shownin FIG. 8, it is also possible to configure the substrate 4′ by usingglobular glass beads or resin beads 4 c as the small pieces and aligningthem on the sticky sheet 5, preferably in a single layer.

In addition, a material having a magnetic property may also be used asthe substrate. For example, it is possible to densely align magneticparticles having a diameter of 1 to 500 μm on the surface of the stickysheet 5, and to use a metal plate of such as a stainless-steel as thesubstrate. By so doing, a treatment can be made more efficient, forexample, by isolating the collected small pieces from a solution withuse of the magnetic force when the small pieces are treated in thesolution. If a material such as a metal which does not allow thetransmission of visible light is used as the substrate, the small pieces4 b, even though these are small, can be clearly observed by eyes at thetime when observing the transmitted beam image of the sticky sheet 5with the optical microscope.

In this case, a magnet can be used as the pickup unit 8. For example, amagnet having approximately the same size as that of the magneticparticle is provided at the tip of the pickup needle 11. Then, the tipof the pickup needle 11 is brought closer to a magnetic particle fromthe opposite side against the sticky sheet 5, by which a desiredmagnetic particle can be held on the tip of the pickup needle 11 andpicked up from the sticky sheet 5 by the magnetic force.

Moreover, in the above-mentioned embodiment, the employed structure issuch that the optical microscope 2 of an erecting type is used and theexpanding stage 6 is provided separately from the optical microscope 2.However, the structure of the cell collecting system 100 is not to belimited to this. For example, an inverted type can be adopted as theoptical microscope 2, and the expanding stage 6 can be installed in theoptical microscope 2.

FIG. 9 shows an example of the structure in which the expanding stage 6and the jig 7 are provided to an inverted-type optical microscope 2. Thespecimen stage 2 b is provided slidably in the horizontal directionalong a guide rail 2 c bridged between the observation position of theoptical microscope 2 and a position out of the main body of the opticalmicroscope 2. In such a structure, the sticky sheet 5 is expanded on theexpanding stage 6 by moving the jig 7 downward in a state where thespecimen stage is arranged in the observation position. Thereafter, thespecimen stage 2 b is slid and the small pieces 4 b are picked up byusing the pickup unit 8 having been provided separately from the opticalmicroscope 2.

FIG. 10 shows an example of the structure in which the expanding stage6, the jig 7, and the pickup unit 8 are provided to an inverted-typeoptical microscope 2. The expanding stage 6 is provided above thespecimen stage 2 b so that the stage can be moved integrally with theobject lens in the vertical direction, and the expanding stage 6 isdesigned to be pushed against the sticky sheet 5 held on the specimenstage 2 b by the jig 7, from the above. In this structure, a series ofoperations from the expanding of the sticky sheet 5 to the pickup of thesmall pieces 4 b can be done on the specimen stage 2 b.

Moreover, in the above-mentioned embodiment, the small pieces 4 b to bepicked up are determined by making a comparison between the analytesection image of the section for pick-up purpose and the stained-sectionimage of the section for staining purpose. However, instead of this, itis also possible to determine the small pieces 4 b to be picked up fromthe image of the section for pick-up purpose.

In the case where a section for pick-up purpose has a relatively thickthickness, cells are overlapped in the thickness direction. This makesit difficult to accurately recognize the shape of each cell, thedistribution of cells, and the like, from the image, even though theimage is stained. However, even from such a stained-section image or anunstained-section image of the section, in the case where thedetermination of the region to be picked up is possible based on a roughshape of the tissue or on the distribution of cells, for example asshown in FIG. 11, the small pieces 4 b to be picked up can be selectedusing the same image while observing the section for pick-up purposewith the optical microscope 2 (Step S11). Thereafter, the sticky sheet 5can be expanded (Step S9) and the small pieces 4 b can be picked up(Step S10). By so doing, the procedure can be much more simplified asrequired.

Moreover, in the above-mentioned embodiment, the approximately columnarexpanding stage 6 is used to expand the sticky sheet 5. However, theunit to expand the sticky sheet 5 is not to be limited to this.

For example, instead of the expanding stage 6, an outer cylinder 14 aand an inner cylinder 14 b that can be accommodated in the outercylinder 14 a can be adopted.

In this case, as shown in FIG. 12, the sticky sheet 5 is covered overthe top surface of the outer cylinder 14 a and fixed to the lateralsurface of the outer cylinder 14 a. The symbol 15 denotes a fixingmember for fixing the sticky sheet 5 to the lateral surface of the outercylinder 14 a. Next, as shown in FIG. 13 the inner cylinder 14 b isprotruded relatively from the inside of the outer cylinder 14 a, or asshown in FIG. 14 the inner cylinder 14 b is pushed relatively from theoutside of the outer cylinder 14 a into the inside of the outer cylinder14 a. By so doing, the sticky sheet 5 can be expanded approximatelyevenly in every direction.

In addition, the structure may also be such that as shown in FIG. 15Athe sticky sheet 5 is held in a certain shape by the holder 16 in aposition where the region adhered with the glass substrate 4 can beseen, and as shown in FIG. 15B the sticky sheet 5 is expanded bypressing the region adhered with the glass substrate 4 against theexpanding stage 6.

Moreover, as for the grip rings, it is also possible to adopt rings thatcan be held in a mutually and tightly attached state by a magneticforce. For example, as shown in FIG. 16, grip rings 10 c and 10 d can becomposed of a top ring 10 c made from a magnet and an bottom ring 10 dmade from a metal such as a stainless-steel having the same diameters,so that the both rings 10 c and 10 d can be tightly attached to eachother by a magnetic force while sandwiching the sticky sheet 5 in aexpanded state between the top ring 10 c and the bottom ring 10 d. By sodoing, the sticky sheet 5 can be held in a expanded state. The bothrings 10 c and 10 d may also be composed of magnets which generateattracting forces each other.

Moreover, in the above-mentioned embodiment, a small piece 4 b isdetached and collected from the sticky sheet 5 by pushing the smallpiece 4 b from the back with the pickup needle 11. However, instead ofthis, it is also possible to use a suction member the interior of whichcan be sucked to a negative pressure. By so doing, a small piece 4 b tobe picked up can be detached from the sticky sheet 5 and collected intothe suction member by bringing the suction member closer to the smallpiece 4 b from the opposite side against the sticky sheet 5.

Moreover, a substance that loses the stickiness by UV irradiation canalso be used as the sticky sheet 5.

In this case, for example, a UV light source is equipped in the opticalmicroscope 2. Also, UV light radiated from the UV light source isirradiated locally to the position of a small piece 4 b to be picked upthrough the object lens, by which the small piece 4 b can be easilydetached and collected from the sticky sheet 5. Besides, a similareffect can also be achieved by guiding UV light through an optical fiberand locally irradiating the light toward the position of the small piece4 b to be picked up while orienting the fiber end thereto.

<Embodiment for Picking Up Small Pieces by Suction>

It is also possible to pick up an optional small piece from the stickysheet by using a tubular suction member in which a hole having a sizenot allowing each small piece to pass through, is made at the tip. Theinterior of the suction member can be set to a negative pressure asrequired, by which the small piece can be adhered at the tip, and thentransferred and picked up from the top of the sticky sheet. It is alsopossible to adopt a form in which a plurality of such tubular suctionmembers are bundled, or to make a plurality of holes having sizes notallowing each small piece to pass through, in an arrangementcorresponding to the spacing between the small pieces in a expandedstate, at the tip of the tubular suction member. By using such a member,a plurality of small pieces can be picked up at the same time.

In addition, it is also possible to make the size of the hole in thetubular suction member larger than each small piece so that small piecescan be collected into the tube by sucking.

Furthermore, it is also possible to set a suction member made from aconductive material. By so doing, the small pieces can be more easilyhandled because electricity can be removed from the small pieces eventhough they have been electrified.

<Embodiment for Picking Up Small Pieces After Detachment of all or aPart of Small Pieces from the Expanded Sticky Sheet>

If a UV-sensitive adhesive is used for the sticky sheet, the stickinesscan be weakened by UV irradiation. It is possible to detach all oroptional small pieces onto the flat plate 18 by a needle assembly member17 as shown in FIG. 19 in a state where the stickiness has been weakenedin such a sticky sheet, and to pick up necessary small pieces from thegroup of the detached small pieces on the flat plate 18. Regarding themethod for picking up, it is possible to pick up by holding each piecewith a pair of tweezers, or to pick up by sucking each piece with theabove-mentioned tubular suction member 19 as shown in FIG. 20A or FIG.20B.

<Embodiment for Picking Up Small Pieces with a Needle or a SuctionMember While Tilting the Expanded Sticky Sheet>

It is also possible to pick up small pieces not by orienting theexpanded sticky sheet vertically downward but by tilting it relative tothe vertical line to drop them. In this case, it is easier to arrange anobservation unit 24 to observe the small pieces and a container 23 forcollecting the dropped small pieces, as compared to the case where thesticky sheet is set horizontal. Regarding the method for picking up, asshown in FIG. 21A, a funnel-shaped guide member 22 can be used to pickup the small pieces having been dropped by pushing from the back of theexpanded sticky sheet 5 with a needle 21. Or, they can be picked up bysucking with the above-mentioned tubular suction member 19 as shown inFIG. 21B.

<Embodiment for Collecting Small Pieces by Arranging a Funnel-ShapedMember Above a Container>

It is also possible to arrange a sufficiently large funnel-shaped memberabove the collection container. By so doing, small pieces can be easilypicked up without dropping out from the collection container even thoughits opening is small.

Example

Next is a description of Example of the above-mentioned embodiment.

In this Example, the following experiment was conducted so as to confirmthat the cell collection apparatus was capable of dividing a section ofa biological tissue.

A pig colon was used as the biological tissue. A section having fourside lengths of about 10 mm and a thickness of 50 μm was cut out fromthe biological tissue by freeze sectioning. The cut out section waspasted on two cover glasses, which had been aligned to be adjacent toeach other at the corners and pasted on a dicing sheet (expandablemember), so that the section was bridged over the border (dividing line)between these cover glasses. Thereafter, the section was dried by air.The cover glass used herein had respective side lengths of 18 mm and athickness of 0.13 to 0.17 mm.

Next, the dicing tape was pasted on the top frame having a circularwindow formed through an approximate center thereof, so that the sectionwas arranged in an approximate center of the window. Next, the surfacepasted with the cover glasses was faced upward. In a state where thecolumnar stage was arranged in the window, the top frame was pushed downwhile being kept approximately horizontal, by which the portion of thedicing tape arranged within the window was expanded in the planardirection. By so doing, these two cover glasses was separated from eachother, and the section pasted on the cover glasses was divided into twofragments along the border between the cover glasses. The photographs ofthe section before and after dividing the section in this way are shownin FIG. 17 and FIG. 18.

FIG. 17 shows a state before expanding the dicing tape. The line L seenin the vertical direction in the approximate middle of the photograph isthe border (dividing line) between the cover glasses.

FIG. 18 shows the state after expanding the dicing tape. It was foundthat the section was sharply divided along the border between the coverglasses.

From the above-mentioned experiment, it was confirmed to be possible, byusing the cell collection apparatus and the cell collecting system ofthe present invention, to easily divide a section sufficiently sharplyalong the dividing line, even though the thickness is relatively thick.

REFERENCE SIGNS LIST

-   1 Cell collection apparatus-   2 Optical microscope (Observation device)-   2 a Camera port-   2 b Specimen stage-   3 Imaging device-   4 Glass substrate (Substrate)-   4 a Groove (Dividing line)-   4 b Small piece-   5 Sticky sheet (Expandable member)-   6 Expanding stage (Expansion unit, Pressing member)-   7 Jig-   8 Pickup unit-   9 a Bottom frame (Fixing member)-   9 b Top frame-   9 c and 9 d Windows-   10 a Grip ring, Outer ring (Holding member)-   10 b Grip ring, Inner ring (Holding member)-   10 c Grip ring, Top ring (Holding member)-   10 d Grip ring, Bottom ring (Holding member)-   11 Pickup needle (Needle member)-   12 Manipulator-   12 a Arm-   13 Tube rack-   13 a Collection tube-   14 a Outer cylinder-   14 b Inner cylinder-   15 Fixing member-   16 Holder-   17 Needle assembly member-   18 Flat plate-   19 Suction member-   21 Needle-   22 Funnel-shaped guide member-   23 Collection container-   24 Observation unit-   100 Cell collecting system

1. A cell collection apparatus comprising: a substrate which is providedto be dividable into a plurality of small pieces along a predetermineddividing line, and which has a flat surface to which a section of abiological tissue can be pasted; a sheet-shaped expandable member towhich the substrate can be adhered in a detachable manner, and which isexpandable in a direction along the surface; a expansion unit forexpanding the expandable member in at least a region adhered with thesubstrate, in a direction along the surface; and a pickup unit fordetaching and picking up the divided small pieces from the expandablemember.
 2. A cell collection apparatus according to claim 1, wherein theexpandable member has stickiness on its surface.
 3. A cell collectionapparatus according to claim 1, wherein the substrate comprises aplurality of separated small pieces adhered to the expandable member inmutually adjacent and aligned state.
 4. A cell collection apparatusaccording to claim 1, wherein the dividing line is composed of a grooveformed in the surface of the substrate.
 5. A cell collection apparatusaccording to claim 1, wherein the expandable member comprises anoptically transparent or semi-transparent material.
 6. A cell collectionapparatus according to claim 1, wherein the small piece is in acuboid-shape having a thickness of 0.05 to 0.5 mm and side lengths of0.05 to 5.0 mm.
 7. A cell collection apparatus according to claim 1,wherein the expansion unit comprises: a fixing member for fixing aperiphery of the region of the expandable member being adhered with thesubstrate, in a predetermined shape; and a pressing member for pressingthe region of the expandable member being fixed by the fixing member,from a side of the surface opposite side to the surface adhered with thesubstrate.
 8. A cell collection apparatus according to claim 1, whereinthe pickup unit comprises: a needle member for pushing a position of theexpandable member being adhered with the small pieces, from the surfaceon the opposite side to the surface adhered with the small pieces.
 9. Acell collection apparatus according to claim 1, wherein the cellcollection apparatus further comprises: a holding member for holding theregion of the expandable member in a expanded state.
 10. A cellcollection apparatus according to claim 9, wherein the holding member iscapable of holding the surface of the expandable member on the sideadhered with the substrate to be faced downward.
 11. A cell collectionapparatus according to claim 1, wherein the pickup unit is a suctionmember which adheres by suction to and picks up the small pieces fromthe surface of the expandable member where the substrate is adhered, andthe interior of which can be sucked to a negative pressure.
 12. A cellcollection apparatus according to claim 3, wherein the small piece is amagnetic particle having a diameter of 0.001 to 0.5 mm.
 13. A cellcollection apparatus according to claim 12, wherein the pickup unit hasa magnet which generates a magnetic force to attract the magneticparticle, in a space of an approximately same size as that of thedimension of the small piece.
 14. A cell collecting system comprising: acell collection apparatus according to claim 1; and an observationdevice for observing the section on the substrate being adhered to theexpandable member.
 15. A cell collecting method comprising: (a) pastinga section of a biological tissue on a surface of a substrate that can bedivided into a plurality of small pieces along a predetermined dividingline, while having the section bridged over the dividing line; (b)dividing the substrate and the section of the biological tissue alongthe dividing line, by expanding the substrate pasted with the section ina direction along the surface; and (c) picking up the small pieces ofthe section that has been divided in the step (b).